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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210105	1/14	Homo sapiens	HeLa	(d)(U)C	Mathieu, Mathilde	2021	78%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ Basigin/ SLC3A2/ Syntenin/ CD9/ CD81 non-EV: Calnexin/ acetylcholinesterase Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1,25E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 200000 Wash: volume per pellet (ml) 6 Wash: time (min) 70 Wash: Rotor Type MLA-80 Wash: speed (g) 200000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ Syntenin/ Basigin/ SLC3A2/ CD81 Detected contaminants acetylcholinesterase Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 70-400 EV concentration Yes Particle yield No NA EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Close-up, Wide-field Report size (nm) 40-300

	EV210105	7/14	Homo sapiens	HeLa	(d)(U)C	Mathieu, Mathilde	2021	78%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin BafA1 Focus vesicles small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ CD81/ CD9/ basigin/ syntenin/ SLC3A2 non-EV: Calnexin/ acetylcholinesterase Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1,25E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 200000 Wash: volume per pellet (ml) 6 Wash: time (min) 70 Wash: Rotor Type MLA-80 Wash: speed (g) 200000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD81/ syntenin/ basigin/ SLC3A2/ CD63 Detected contaminants Calnexin/ acetylcholinesterase Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 70-400 EV concentration Yes Particle yield No NA EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 40-300

	EV210105	2/14	Homo sapiens	HeLa	(d)(U)C	Mathieu, Mathilde	2021	56%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles large extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ Basigin/ SLC3A2/ Syntenin/ CD9/ CD81 non-EV: Calnexin/ acetylcholinesterase Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1,25E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 40 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 6 Wash: time (min) 20 Wash: Rotor Type MLA-80 Wash: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ SLC3A2/ CD81 Not detected EV-associated proteins CD63/ Syntenin/ Basigin Not detected contaminants Calnexin/ acetylcholinesterase Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 70-400 EV concentration Yes Particle yield No NA

	EV210105	3/14	Homo sapiens	HeLa	(d)(U)C	Mathieu, Mathilde	2021	56%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles large extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ Basigin/ SLC3A2/ Syntenin/ CD9/ CD81 non-EV: Calnexin/ acetylcholinesterase Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1,25E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 20 Pelleting: rotor type Eppendorf Centrifugeuses 5810R Pelleting: speed (g) 2000 Wash: volume per pellet (ml) 50 Wash: time (min) 20 Wash: Rotor Type Not specified;MLA-80 Wash: speed (g) 2000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ SLC3A2 Not detected EV-associated proteins CD63/ Syntenin/ Basigin/ CD81 Not detected contaminants Calnexin/ acetylcholinesterase Characterization: Lipid analysis No

	EV210105	8/14	Homo sapiens	HeLa	(d)(U)C	Mathieu, Mathilde	2021	56%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin BafA1 Focus vesicles large extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ CD81/ CD9/ basigin/ syntenin/ SLC3A2 non-EV: Calnexin/ acetylcholinesterase Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1,25E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 40 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 6 Wash: time (min) 20 Wash: Rotor Type MLA-80 Wash: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD81/ basigin/ SLC3A2 Not detected EV-associated proteins syntenin/ CD63/ lamp1 Not detected contaminants Calnexin/ acetylcholinesterase Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 70-400 EV concentration Yes Particle yield No NA EM

	EV210105	9/14	Homo sapiens	HeLa	(d)(U)C	Mathieu, Mathilde	2021	56%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin GW4869 Focus vesicles small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1,25E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 200000 Wash: volume per pellet (ml) 6 Wash: time (min) 70 Wash: Rotor Type MLA-80 Wash: speed (g) 200000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 70-400 EV concentration Yes Particle yield No NA EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 40-300

	EV210105	12/14	Homo sapiens	HeLa	(d)(U)C UF IAF qEV	Mathieu, Mathilde	2021	38%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 38% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF IAF qEV Protein markers EV: CD9 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Commercial kit qEV Immunoaffinity capture Selected surface protein(s) GFP Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 110 EV concentration Yes Particle yield number of particles per cell;Yes, other: 100

	EV210105	13/14	Homo sapiens	HeLa	(d)(U)C UF IAF qEV	Mathieu, Mathilde	2021	38%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 38% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin RUSH CD63-GFP Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF IAF qEV Protein markers EV: GFP/ CD9 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Commercial kit qEV Immunoaffinity capture Selected surface protein(s) GFP Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ GFP Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 110 EV concentration Yes Particle yield number of particles per cell;Yes, other: 1000

	EV210105	14/14	Homo sapiens	HeLa	(d)(U)C UF IAF qEV	Mathieu, Mathilde	2021	38%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 38% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin RUSH CD9-GFP Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF IAF qEV Protein markers EV: GFP/ CD9 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Commercial kit qEV Immunoaffinity capture Selected surface protein(s) GFP Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins GFP/ CD9 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 110 EV concentration Yes Particle yield number of particles per cell;Yes, other: 500

	EV210105	4/14	Homo sapiens	HeLa	(d)(U)C	Mathieu, Mathilde	2021	34%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 34% (78th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin RUSH plasmids transfection (CD63-Cherry, CD63-YA-Cherry) Focus vesicles small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ Basigin/ SCL3A2/ CD81/ Syntenin/ CD9 non-EV: Calnexin/ acetylcholinesterase Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1,25E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 200000 Wash: volume per pellet (ml) 6 Wash: time (min) 70 Wash: Rotor Type MLA-80 Wash: speed (g) 200000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63 Characterization: Lipid analysis No

	EV210105	5/14	Homo sapiens	HeLa	(d)(U)C	Mathieu, Mathilde	2021	34%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 34% (78th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin RUSH plasmids transfection (CD63-Cherry, CD63-YA-Cherry) Focus vesicles large extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ Basigin/ SCL3A2/ CD81/ Syntenin/ CD9 non-EV: Calnexin/ acetylcholinesterase Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1,25E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 40 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 6 Wash: time (min) 20 Wash: Rotor Type MLA-80 Wash: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9 Not detected EV-associated proteins CD63 Characterization: Lipid analysis No

	EV210105	6/14	Homo sapiens	HeLa	(d)(U)C	Mathieu, Mathilde	2021	34%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 34% (78th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin RUSH plasmids transfection (CD63-Cherry, CD63-YA-Cherry) Focus vesicles large extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ Basigin/ SCL3A2/ CD81/ Syntenin/ CD9 non-EV: Calnexin/ acetylcholinesterase Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1,25E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 20 Pelleting: rotor type Eppendorf Centrifugeuses 5810R Pelleting: speed (g) 2000 Wash: volume per pellet (ml) 50 Wash: time (min) 20 Wash: Rotor Type Not specified;MLA-80 Wash: speed (g) 2000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9 Not detected EV-associated proteins CD63 Characterization: Lipid analysis No

	EV210105	10/14	Homo sapiens	HeLa	(d)(U)C	Mathieu, Mathilde	2021	34%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 34% (78th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin GW4869 Focus vesicles large extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1,25E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 40 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 6 Wash: time (min) 20 Wash: Rotor Type MLA-80 Wash: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 70-400 EV concentration Yes Particle yield No NA EM

	EV210105	11/14	Homo sapiens	HeLa	(d)(U)C UF IAF	Mathieu, Mathilde	2021	25%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 25% (63rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF IAF Protein markers EV: CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Immunoaffinity capture Selected surface protein(s) CD63, CD9 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV210105
species	Homo sapiens
sample type	Cell culture
cell type	HeLa
medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	Serum free medium	Serum free medium	Serum free medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	Serum free medium
condition	Control condition	BafA1	Control condition	Control condition	BafA1	GW4869	Control condition	RUSH CD63-GFP	RUSH CD9-GFP	RUSH plasmids transfection (CD63-Cherry CD63-YA-Cherry)	RUSH plasmids transfection (CD63-Cherry CD63-YA-Cherry)	RUSH plasmids transfection (CD63-Cherry CD63-YA-Cherry)	GW4869	Control condition
separation protocol	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C UF IAF qEV	(d)(U)C UF IAF qEV	(d)(U)C UF IAF qEV	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C UF IAF
vesicle related term	small EVs	small EVs	large EVs	large EVs	large EVs	small EVs	EV	EV	EV	small EVs	large EVs	large EVs	large EVs	EV
Exp. nr.	1	7	2	3	8	9	12	13	14	4	5	6	10	11
EV-METRIC %	78	78	56	56	56	56	38	38	38	34	34	34	34	25

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